Analog of haemophilus Hin47 with reduced protease activity

ABSTRACT

An isolated and purified analog of Haemophilus influenzae Hin47 protein has a decreased protease activity which is less than about 10% of that of natural Hin47 protein and preferably substantially the same immunogenic properties as natural Hin47 protein. An isolated an purified nucleic acid molecule encoding the Hin47 analog may be provided in a recombinant plasmid which may be introduced into a cell which is grown to produce the Hin47 analog. Immunogenic compositions comprising the Hin47 analog and the encoding nucleic acid may be formulated as vaccines for in vivo administration to a host, including a human, to confer protection against diseases caused by a bacterial pathogen, including Haemophilus species, such as Haemophilus influenzae, that produces Hin47 protein or a protein capable of inducing antibodies in the host specifically reactive with Hin47 protein. The Hin47 analog and the encoding nucleic acid also may be employed in diagnostic applications.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with immunogens and antigens from species ofHaemophilus.

BACKGROUND OF THE INVENTION

Haemophilus influenzae is the organism responsible for a variety ofserious human diseases, such as meningitis, epiglotitis, pneumonia andotitis media. Haemophilus influenzae type b (Hib) is a major cause ofbacterial meningitis in children under the age of five years. Protectiveantibodies to the disease are induced by the capsular polysaccharide ofthe organism and vaccines have been developed that utilise the purifiedpolyribosyl ribotol phosphate (PRP) as the antigen. This vaccineprovides 90% protection in adults and in children over 24 months of age,but was ineffective in children under 24 months (Zangwill et al 1993)(The references are identified in a list of references at the end ofthis disclosure, each of which reference in the list is herebyincorporated by reference without further reference thereto). Like otherpolysaccharide antigens, PRP does not induce the proliferation ofT-helper cells, and re-immunisation fails to elicit either a boosterresponse or an increase in memory cells. Conjugation of the PRPpolysaccharide with protein carriers confers T-cell dependentcharacteristics to the vaccine and substantially enhances theimmunologic response to the PRP antigen. Currently, there are fourPRP-carrier conjugate vaccines available. These are vaccines based uponH. influenzae type b capsular polysaccharide conjugated to diphtheriatoxoid, tetanus toxoid, or Neisseria meningitidis outer membrane protein(reviewed in Zangwill et al 1993). These H. influenzae b conjugatevaccines have dramatically reduced the incidence of bacterial meningitis(Schoendorf et al., 1994).

There are six serotypes of H. influenzae designated a to f, which aredefined by their capsular polysaccharides. The current Haemophilusconjugate vaccines do not protect against other invasive typable strains(types a and c) and, importantly, do not protect against non-typable(NTHi) strains which are a common cause of postpartum and neonatalsepsis, pneumonia and otitis media. Otitis media is the most commonillness of early childhood with approximately 70% of all childrensuffering at least one bout of otitis media before the age of seven.Chronic otitis media can lead to hearing, speech, and cognitiveimpairment in children. It is caused by bacterial infection withStreptococcus pneumoniae (approximately 50%), non-typable H. influenzae(approximately 30%), and Moraxella (Branhamella) catarrhalis(approximately 20%). In the United States alone, treatment of otitismedia costs between 1 and 2 billion dollars per year for antibiotics andsurgical procedures, such as tonsillectomies, adenoidectomies andinsertion of tympanostomy tubes. To achieve universal protection againstH. influenzae related diseases, particularly in the two to six month agegroup and certain high risk groups, the provision of conserved,cross-reactive non-capsular H. influenzae immunogens is desirable.Non-typable strains of H. influenzae are also important pathogensresponsible for pneumonia in the elderly and other individuals who areparticularly susceptible to respiratory infections. There is thus a needfor antigens from H. influenzae which are useful as components inimmunogenic preparations that provide protection against the manyserotypes of H. influenzae, PCT application WO 92/10936, published Jul.9, 1992 and incorporated herein by reference thereto, describes a 47,000molecular weight outer membrane protein obtained from H. influenzae thatis reported to be an adhesin and has been termed Hin47 that isimmunologically conserved between non-typable, type b and non-typedclinical isolates of H. influenzae. The amino acid sequence of Hin47 andthe nucleotide sequence of the gene encoding Hin47 were presented at theAmerican Society of Microbiology (ASM) conference held in New Orleans,May 26-30, 1992. These sequences have also been published in PCTapplication WO 94/00149, published Jan. 6, 1994 and incorporated hereinby reference thereto.

Since Hin47 is conserved among strains of Haemophilus influenzae, and isreported to be an adhesin, the protein has utility in diagnosis of andvaccination against disease caused by H. influenzae or other bacterialpathogens that produce Hin47 or a protein capable of raising antibodiesspecifically reactive with Hin47.

A disadvantage of Hin47 for use as an antigen in diagnosis, for thegeneration of anti-Hin47 antibodies useful in diagnosis and as animmunogen in vaccination is the unexpected discovery by the presentapplicants that Hin47 has protease activity which results in theautodigestion of Hin47 and the proteolytic degradation of other antigensmixed therewith.

It would be advantageous to provide analogs of Hin47 protein (sometimesreferred to herein as mutants or derivatives) that are substantiallyreduced in proteolytic activity for use as antigens, immunogenicpreparations including vaccines, carriers for other immunogens and thegeneration of diagnostic reagents.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision of analogs ofHaemophilus Hin47 protein having reduced protease activity.

In accordance with one aspect of the invention there is provided anisolated and purified analog of Haemophilus influenzae Hin47 proteinhaving a decreased protease activity which is less than about 10% ofnatural Hin47 protein. Such Hin47 analog preferably has substantiallythe same immunogenic properties of natural Hin47 protein. The analog ofthe present invention may be produced by chemical, biochemical orgenetic modification of natural Hin47.

In one embodiment of the present invention, when the analog is producedby genetic modification, at least one amino acid of the natural Hin47contributing to protease activity may be deleted or replaced by adifferent amino acid to produce the reduced protease activity.Alternatively, the reduced protease activity may be achieved byinserting at least one amino acid into the natural Hin47 protein. The atleast one deleted or replaced amino acid may be selected from aminoacids 195 to 201 of Hin47, and specifically may be Serine-197, which maybe deleted or replaced by alanine. In addition, the at least one deletedor replaced amino acid may be His-91 and may be deleted or replaced byalanine or lysine or arginine. Further, the at least one deleted orreplaced amino acid may be Asp-121 and may be deleted or replaced byalanine or glutamic acid.

In a further aspect, the present invention provides an isolated andpurified nucleic acid molecule comprising a mutant Haemophilusinfluenzae hin47 gene encoding an analog of Haemophilus influenzae Hin47protein having a reduced protease activity which is less than about 10%of natural Hin47 protein. The mutant hin47 gene may encode any of theHin47 analogs discussed above. The mutant gene preferably is formed bysite-directed mutagenesis of a wild-type hin47 gene. The nucleic acidmolecule may be contained in a recombinant plasmid adapted fortransformation of a host and may be plasmid DS-1011-1-1 (deposited onJul. 27, 1994 at American type Culture Collection under Accession No.75845. The invention also includes a transformed cell containing such arecombinant plasmid.

The present invention, in another aspect, includes a method forproducing an analog of Haemophilus influenzae Hin47 protein having areduced protease activity which is less than about 10% of natural Hin47protein, which comprises identifying at least one amino acid residue ofHin47 protein which contributes to protease activity thereof, effectingsite-directed mutagenesis of the hin47 gene to remove or replace anucleotide sequence encoding the at least one amino acid and to producea mutated hin47 gene, introducing the mutated hin47 gene into a cell toproduce a transformed cell and growing the transformed cell to producethe Hin47 analog. The at least one amino acid which is selected may beany of the ones specifically identified above with respect to the Hin47analog.

The introduction of the mutated hin47 gene preferably produces atransformed cell in which the mutated hin47 gene is under control of theT7 promoter and the growing of the transformed cell and expression ofthe Hin47 analog of the T7 promoter than preferably is effected byculturing in an inducing concentration of lactose. Preferably, theintroduction of the mutated hin47 is effected by transforming the cellwith the recombinant plasmid "DS-1011-1-1, sometimes otherwise referredto as plasmid" pT7/Hin47.

A further aspect of the invention provides a method of providingisolated and purified Hin47 analog, which comprises effecting theprocedure described above for the production of the Hin47 analog toproduce grown transformed cells harbouring inclusion bodies containingthe Hin47 analog, disrupting the grown transformed cells to producesupernatant and the inclusion bodies, solubilizing the inclusion bodiesto produce a solution containing Hin47 analog, chromatographicallypurifying the Hin47 analog from the solution free from cell debris, andisolating the purified Hin47 analog.

The analogs of Hin47 provided herein with their decreased proteolyticactivity are useful as antigens in immunogenic composition, carriers forother immunogens, diagnostic agents and in the generation of diagnosticagents. The nucleic acid molecules also are useful as probes fordiagnostic use and also as in immunogenic compositions.

In a further aspect of the invention, there is provided an immunogeniccomposition comprising an immuno-effective amount of the Hin47 analog orof the nucleic acid molecule including the gene encoding the Hin47analog. The immunogenic composition may be formulated as a vaccine forin vivo administration to a host, including a human, to conferprotection against diseases caused by a bacterial pathogen that producesHin47 or a protein capable of inducing antibodies in the hostspecifically reactive with Hin47. The bacterial pathogen may be aHaemophilus species, such as Haemophilus influenzae. The immunogeniccompositions of the invention may further comprise at least one otherimmunogenic or immunostimulating material, such as an adjuvant. In anadditional embodiment, the nucleic acid molecule comprising a geneencoding the Hin47 analog may be contained within a live vector, such asa pox virus, Salmonella, poliovirus, adenovirus, vaccinia or BCG.

The invention also extends to a method of generating an immune responsein a host, including a human, comprising administering thereto animmuno-effective amount of the immunogenic compositions provided herein.

As mentioned above, the Hin47 analog provided herein is useful indiagnostic applications. Accordingly, in an additional aspect of theinvention, there is provided a method of determining the presence ofantibodies specifically reactive with Hin47 in a sample, comprising thesteps of:

(a) contacting the sample with the Hin47 analog having substantially thesame immunogenic properties as the natural Hin47 protein as providedherein to produce complexes comprising the Hin47 analog and any suchantibodies present in the sample specifically reactive therewith; and

(b) determining the production of the complexes.

The present invention also provides a method of determining the presenceof Hin47 in a sample, comprising the steps of:

(a) immunizing a subject with an immunogenic composition as providedherein to produce antibodies specific for Hin47 protein;

(b) contacting the sample with the antibodies to produce complexescomprising any Hin47 present in the sample and the Hin47 specificantibodies; and

(c) determining production of the complexes.

The invention also extends to a diagnostic kit for determining thepresence of antibodies in a sample specifically reactive with Hin47,comprising:

(a) the Hin47 analog having substantially the same immunogenicproperties as the natural Hin47 protein as provided herein;

(b) means for contacting the analog with the sample to produce a complexcomprising the analog and any such antibodies present in the sample; and

(c) means for determining production of the complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the restriction maps of plasmids JB-1031-1-14 andJB-1068-2-2 -and the construction of the plasmids for sequence analysis;

FIG. 2 shows the full nucleotide (SEQ ID NO: 1) and deduced amino acidsequence (SEQ ID NO: 2) of Hin47 from H. influenzae strain SB33 as wellas a partial nucleotide sequence (SEQ ID NO: 3) and a partial deducedamino acid sequence (SEQ ID NO: 4) thereof, the latter beingspecifically copied by an inventor herein from materials presented inthe ASM conference as described above;

FIG. 3 shows a comparison of the amino acid sequences of H. influenzaeHin47 (SEQ ID NO: 2), E. coli htrA (SEQ ID NO: 5), and Salmonellatyphimurium htrA (SEQ ID NO: 6);

FIG. 4 shows an alignment of amino acid residues 57 to 256 of Hin47 withcertain known proteases (SEQ ID NOS: 7 to 16). Codes are as follows:TON, rat tonin; PKAAB, kallikrein; PTN, trypsin; CHAA, chymotrypsin;EST, elastase; RP2A, rat mast cell protease; SGT, Streptomyces griseustrypsin; SGBE, S. griseus proteinase A; SGA, S. griseus proteinase B;ALP, L. enzymogenes alpha-lytic protease; hin47, res. 57-256 of Hin47.Asterisks (*) denote structurally conserved regions. The catalytic triadresidues are indicated by a hash mark (#). `con` refers to regions ofstructural concensus, among the mammalian proteases;

FIG. 5 shows the restriction maps for plasmids DS-1011-1-1 and DS-1048-2which express a Hin47 analog from E. coli and a construction scheme forplasmid DS-1011-1-1(pT7/Hin47*);

FIG. 6 shows a process for purifying the Hin47 analog from E. coliaccording to one embodiment of the present invention and gel analysis ofthe purified product;

FIG. 7 shows the protease activities of natural Hin47 and Hin47 analogtowards β-casein;

FIG. 8 shows the stability of natural Hin47 and the Hin47 analog atdifferent temperatures;

FIG. 9 shows the enzymatic degradation of an H. influenzae recombinantprotein by natural Hin47 and the Hin47 analog; and

FIG. 10 shows the comparative immunogenicity of natural Hin47 and theHin47 analog in mice.

GENERAL DESCRIPTION OF INVENTION

Any Haemophilus strains that have Hin47 genes may be conveniently usedto provide the purified and isolated nucleic acid molecules (which maybe in the form of DNA molecules), comprising at least a portion codingfor Hin47 as typified by embodiments of the present invention. Suchstrains are generally available from clinical sources and from bacterialculture collections, such as the American Type Culture collection. Suchstrains include H. influenzae strains and other bacteria that produce aprotein capable of generating antibodies that specifically recognizeHin47 fragment or analog thereof. Appropriate strains of Haemophilus mayinclude:

H. influenzae type b strain MinnA;

H. influenzae type b strain Eagan;

H. influenzae non-typable strain SB33; or

H. influenzae non-typable strain PAK 12085.

Referring to FIG. 1, there is illustrated restriction maps of plasmidsJB-1031-1-14 and JB-1068-2-2 that contain a portion encoding Hin47protein from non-typable H. influenzae SB33. The nucleotide sequence ofthe Hin47 gene was determined and is shown in FIG. 2 along with thededuced amino acid sequence of the Hin47 protein. Referring to FIG. 3,there is shown an amino acid sequence alignment of H. influenzae Hin47and the serine proteases htrA from Escherichia coli and htrA fromSalmonella typhimurium. This alignment for the first time reveals theunexpected discovery of the present applicants that Hin47 is related tobacterial serine proteases and that Hin47 has protease activity. Hin47has previously been reported to be an adhesin. The discovered proteaseactivity thereof greatly limits the usefulness of natural Hin47 as animmunogen for vaccination and as an antigen in diagnostic uses. Thesequence alignment shown in FIG. 3 revealed that the htrA proteins andHin47 contain a GNSGGAL (SEQ ID NO: 17) sequence between residues 195and 201 of the mature protein. The consensus sequence of the active siteof serine proteases is GDSGGPK (SEQ ID NO: 18) (Brenner, 1988) and theactive residue is serine. Thus, Serine-197 in Hin47 was mutated toproduce an analog of Hin47 reduced in protease activity, in accordancewith one embodiment of the invention. In a particular embodiment,Serine-197 was replaced by alanine. Amino acid residues 57 to 256 ofHin47 were further aligned with known proteases and the active siteresidues identified from the local homologies surrounding the residuesof the catalytic triad (FIG. 4). There is a standard numbering systemfor serine proteases in which the catalytic triad residues are numberedas His-57, Asp-102 and Ser-195. These correspond to residues His-91,Asp-121 and Ser-197 in the sequential numbering system. Thus, referringto FIG. 4, there is shown a structure-based alignment of tenstructurally determined serine proteases (SEQ ID NOS: 7 to 16) in whichhomologous residues are aligned primarily on the basis of similarlocations in three-dimensional space. The location of many of theresidues in the hydrophobic core of Hin47, as well as residues aroundthe active site can be aligned reasonably well to identify functionalamino acids of the Hin47 protease. Thus, other amino acid residues inHin47 that contribute to protease activity of the protein include His-91and Asp-121. In particular embodiments, His-91 may be replaced byalanine, lysine or arginine. In an additional embodiment, Asp-121 may bereplaced by alanine or glutamic acid. Although the provision of ananalog of Hin47 having reduced protease activity has been exemplifiedherein by particular amino acid substitution within Hin47 protein, thediscovery of the protease activity and the methods of Hin47 expression,purification and analysis provided herein, allow for the production ofother analogs having at least one other amino acid deleted or replacedor having at least one additional amino acid inserted into the Hin47protein. In particular applications and embodiments, it may be desirableto simultaneously alter several amino acids of the Hin47 protein toparticularly reduce the protease activity of Hin47. Accordingly, thepresent invention provides analogs of Hin47 protein having decreasedprotease activity due to single or multiple amino acid deletions,replacements or additions within the Hin47 protein.

Referring to FIG. 5, there is illustrated plasmids DS-1011-1-1 andDS-1048-2 which express a Hin47 analog serine-197→alanine in E. coli.FIG. 6 shows a flow diagram of a method for the purification of theHin47 analog from E. coli inclusion bodies.

FIG. 7 shows the reduced protease activity of the Hin47serine-197→alanine analog on the substrate β-casein and demonstrates theanalog to have less than about 10% of the protease activity of naturalHin47 protein. Thus, in one embodiment of the invention, there isprovided an analog of Hin47 having a protease activity of less thanabout 10% of the protease activity of natural Hin47 and such analog mayspecifically have amino acid Serine-197 replaced by alanine.

Referring to FIG. 8, there is illustrated an analysis of the increasedstability of an analog of Hin47 as provided herein. Thus, in oneembodiment of the present invention, there is provided an analog ofHin47 protein having increased thermal stability, and such analog mayspecifically have amino acid serine-197 replaced by alanine.

Referring to FIG. 9, there is illustrated the proteolytic degradation ofa non-Hin47 Haemophilus antigen by Hin47 and a Hin47 analog as providedherein. Thus, in accordance with a further embodiment of the presentinvention, there is provided an analog of Hin47 compatible with a secondnon-Hin47 protein and such analog may specifically have amino acidSerine-197 replaced by alanine.

Referring to FIG. 10 and Table 1, there is illustrated the comparativeimmunogenicity of unmodified Hin47 and a Hin47 analog having reducedprotease activity in mice. The Hin47 protein and Hin47 analog hadcomparable immunogenicity. Thus, in a particular embodiment, there isprovided an analog of Hin47 having reduced protease activity and havingsubstantially the same immunogenic properties of natural Hin47 protein.Such analog may specifically have amino acid Serine-197 replaced byalanine.

Referring to Table 2, there is shown the immunoprotective properties ofan analog of Hin47 having reduced protease activity against Hib in theinfant rat model of bacteraemia, according to an embodiment of theinvention and such analog may specifically have amino acid Serine-197replaced by alanine.

In accordance with another aspect of the present invention, there isprovided a vaccine against Haemophilus or other bacterial pathogens thatproduce Hin47 or a protein capable of inducing antibodies thatspecifically recognize Hin47, comprising an immunogenically-effectiveamount of an immunoprotective analog of Hin47 as provided herein or anucleic acid molecule having a sequence encoding a Hin47 analog asprovided herein, and a physiologically-acceptable carrier therefor. Theprovided analogs also may be used as a carrier protein for hapten,polysaccharides or peptides to make a conjugate vaccine againstantigenic determinants unrelated to Hin47.

As will be apparent from the following disclosure, the present inventionfurther provides plasmids and novel strains of bacteria for productionof Hin47 analogs as provided herein.

The purified and isolated DNA molecules comprising at least a portioncoding for an analog of Haemophilus influenzae Hin47 protein havingreduced protease activity compared to natural Hin47 typified by theembodiments described herein, are advantageous as nucleic acid probesfor the specific identification of Haemophilus strains in vitro or invivo. The Hin47 analogs encoded by the DNA molecules provided herein areuseful as diagnostic reagents as antigens or for the generation ofanti-Hin47 antibodies, antigens for the vaccination against the diseasescaused by species of Haemophilus and other bacterial pathogens thatproduce a protein capable of producing antibodies that specificallyrecognise Hin47 and for detecting infection by Haemophilus and othersuch bacteria.

In additional embodiments of the present invention, the Hin47 analogshaving reduced protease activity as provided herein may be used ascarrier molecules to prepare chimeric molecules and conjugate vaccines(including glycoconjugates) against pathogenic bacteria, includingencapsulated bacteria. Thus, for example, glycoconjugates of the presentinventions may be applied to vaccinations to confer protection againstdisease and infection caused by any bacteria having polysaccharideantigens including lipooligosaccharides (LOS) and PRP. Bacterialpathogens may include, for example, Haemophilus influenzae,Streptococcus pneumoniae, Escherichia coli, Neisseria meningitidis,Salmonella typhi, Streptococcus mutans, Cryptococcus neoformans,Klebsiella, Staphylococcus aureus and Pseudomonas aeruginosa. Particularantigens which can be conjugated to analogs of Hin47 and methods toachieve such conjugations are described in applicants published PCTapplication WO 94/12641 which is hereby incorporated by referencethereto.

In another embodiment, the carrier function of Hin47 analogs may beused, for example, to induce immunity toward abnormal polysaccharides oftumor cells, or to produce anti-tumor antibodies that can be conjugatedto chemotherapeutic or biocative agents.

Accordingly, the present invention provides the primary sequence and thepreparation of an analog of Hin47 of H. influenzae that can be used inthe prevention and diagnosis of diseases caused by Haemophilus. Inparticular, the inventors discovered that the Hin47 analog can elicitprotective immune response against live H. influenzae type b bacterialchallenge. Thus, the present inventions have utility in vaccines. Theinvention also discloses the nucleotide sequences of the gene encodingthe Hin47 analog. These DNA segments may be used to provide an immunogenessentially free from other H. influenzae antigens, such as PRP andlipooligosaccharides (LOS), through the application of recombinant DNAtechnology. The Hin47 analog protein, may be produced in a suitableexpression system, such as E. coli, Haemophilus, Bacillus, BordetellaFungi, Yeast Baculovirus, Poxvirus, vaccinia or mammalian expressionsystems. The present disclosure further provides novel techniques whichcan be employed for preparing essentially pure Hin47 analogs.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of, for example, Haemophilusinfections, and infections with other bacterial pathogens that produceproteins capable of producing antibodies that specifically recognizeHin47 and the generation of immunological reagents. A furthernon-limiting discussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from Hin47 analogs as disclosed herein. The vaccine elicits animmune response in a subject which produces antibodies, includinganti-Hin47 antibodies and antibodies that are opsonizing orbactericidal. Should the vaccinated subject be challenged by Haemophilusor other bacteria that produce proteins capable of producing antibodiesthat specifically recognize Hin47, the antibodies bind to and inactivatethe bacterium. Furthermore, opsonizing or bactericidal anti-Hin47antibodies may also provide protection by alternative mechanisms.

Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The Hin47 analogs may bemixed with pharmaceutically acceptable excipients which are compatiblewith the Hin47 analog. Such excipients may include, water, saline,dextrose, glycerol, ethanol, and combinations thereof. The immunogeniccompositions and vaccines may further contain auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness thereof. Methods of achieving adjuvant effectinclude the use of agents such as aluminum hydroxide or phosphate(alum), commonly used as 0.05 to 0.1 percent solution in phosphatebuffered saline. Immunogenic compositions and vaccines may beadministered parenterally, by injection subcutaneously orintromuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. Alternatively, othermodes of administration including suppositories and oral formulationsmay be desirable. For suppositories, binders and carriers may include,for example, polyalkalene glycols or triglycerides. Oral formulationsmay include normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and containabout 1 to 95% of the Hin47 analogs. The immunogenic preparations andvaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art and may be of the order of micrograms of theHin47 analogs. Suitable regimes for initial administration and boosterdoses are also variable, but may include an initial administrationfollowed by subsequent administrations. The dosage may also depend onthe route of administration and will vary according to the size of thehost.

The concentration of antigen in an immunogenic composition according tothe invention is in general about 1 to 95%. A vaccine which containsantigenic material of only one pathogen is a monovalent vaccine.Vaccines which contain antigenic material of several pathogens arecombined vaccines and also belong to the present invention. Suchcombined vaccines contain, for example, material from various pathogensor from various strains of the same pathogen, or from combinations ofvarious pathogens.

The nucleic acid molecules encoding the Hin47 analog of the presentinvention may also be used directly for immunization by administrationof the DNA directly, for example, by injection for genetic immunizationor by constructing a live vector, such as Salmonella, BCG, adenovirus,poxvirus, vaccinia or poliovirus. A discussion of some live vectors thathave been used to carry heterologous antigens to the immune system arediscussed in, for example, O'Hagen (1992). Processes for the directinjection of DNA into test subjects for genetic immunization aredescribed in, for example, Ulmer et al., 1993.

2. Immunoassays

The Hin47 analogs of the present invention are useful as immunogens forthe generation of anti-Hin47 antibodies, as antigens in immunoassaysincluding enzyme-linked immunosorbent assays (ELISA), RIAs and othernon-enzyme linked antibody binding assays or procedures known in the artfor the detection of anti-bacterial, Haemophilus, and anti-Hin47antibodies. In ELISA assays, the Hin47 analogs, are immobilized onto aselected surface, for example, a surface capable of binding proteinssuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed Hin47 analogs, a nonspecific protein suchas a solution of bovine serum albumin (BSA) that is known to beantigenically neutral with regard to the test sample may be bound to theselected surface. This allows for blocking of nonspecific adsorptionsites on the immobilizing surface and thus reduces the background causedby nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conductive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from 2 to 4 hours, at temperatures suchas of the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween or a borate buffer. Following formation of specificimmunocomplexes between the test sample and the bound Hin47 analogs, andsubsequent washing, the occurrence, and even amount, of immunocomplexformation may be determined by subjecting the immunocomplex to a secondantibody having specificity for the first antibody. If the test sampleis of human origin, the second antibody is an antibody havingspecificity for human immunoglobulins and in general IgG. To providedetecting means, the second antibody may have an associated activitysuch as an enzymatic activity that will generate, for example, a colordevelopment upon incubating with an appropriate chromogenic substrate.Quantification may then be achieved by measuring the degree of colourgeneration using, for example, a visible spectra spectrophotometer.

3. Use of Sequences as Hybridization Probes

The nucleic aid molecules of the present invention, having the sequenceof the hin47 analog gene, allow for the identification and cloning ofthe Hin47 genes from any species of Haemophilus and other bacteria thatproduce proteins capable of producing antibodies that specificallyrecognize Hin47.

The nucleic acid molecules having the sequence encoding the Hin47 analogof the present invention are useful for their ability to selectivelyform duplex molecules with complementary stretches of other hin47 genes.Depending on the application, a variety of hybridization conditions maybe employed to achieve varying degrees of selectivity of the probetoward the other hin47 genes. For a high degree of selectivity,relatively stringent conditions are used to form the duplexes, such aslow salt and/or high temperature conditions, such as provided by 0.02Mto 0.15M NaCl at temperatures of between about 50° to 70° C. For someapplications, less stringent hybridization conditions are required, suchas 0.15M to 0.9M salt, at temperatures ranging from between about 20° C.to 55° C. Hybridization conditions can also be rendered more stringentby the addition of increasing amounts of formamide, to destabilize thehybrid duplex. Thus, particular hybridization conditions can be readilymanipulated, and will generally be a method of choice depending on thedesired results.

in a clinical diagnostic embodiment, the nucleic acid molecules encodingthe hin47 genes of the present invention may be used in combination withan appropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of providing a detectable signal. Insome diagnostic embodiments, an enzyme tag, such as urease, alkalinephosphatase or peroxidase, instead of a radioactive tag may be used. Inthe case of enzyme tags, colorimetric indicator substrates are knownwhich can be employed to provide a means visible to the human eye orspectrophotometrically, to identify specific hybridization with samplescontaining hin47 gene sequences.

The nucleic acid molecules comprising hin47 genes of the presentinvention are useful as hybridization probes in solution hybridizationsand in embodiments employing solid-phase procedures. In embodimentsinvolving solid-phase procedures, the test DNA (or RNA) from samples,such as clinical samples, including exudates, body fluids (e.g., serum,amniotic fluid, middle ear effusion, sputum, bronchoalveolar lavagefluid) or even tissues, is adsorbed or otherwise affixed to a selectedmatrix or surface. The fixed, single-stranded nucleic acid is thensubjected to specific hybridization with selected probes comprising thenucleic acid sequences of the hin47 genes of the present invention underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria requireddepending on, for example, the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe etc. Followingwashing of the hybridization surface so as to remove non-specificallybound probe molecules, specific hybridization is detected, or evenquantified, by means of the label.

4. Expression of the Genes encoding analogs of Hin47 having reducedprotease activity

Vectors perhaps containing replicon and control sequences which arederived from species compatible with the host cell may be used for theexpression of the Hin47 analog genes as provided herein in expressionsystems. The vector ordinarily carries a replication site, as well asmarking sequences which are capable of providing phenotypic selection intransformed cells. For example, E. coli may be transformed using pBR322which contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage must also contain, or bemodified to contain, promoters which can be used by the host cell forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host can be used as a transforming vectorin connection with these hosts. For example, the phage in lambda GEM™-11may be utilized in making recombinant phage vectors which can be used totransform host cells, such as E. coli LE392.

Promoters commonly used in recombinant DNA construction include theβ-lactamase (penicillinase) and lactose promoter systems (Chang et al.,1979; Goeddel et al., 1980) and other microbial promoters, such as theT7 promoter system (U.S. Pat. No. 4,952,496). Details concerning thenucleotide sequences of promoters are known, enabling a skilled workerto ligate them functionally with plasmid vectors. The particularpromoter used will generally be a matter of choice depending upon thedesired results. Hosts that are appropriate for expression of the Hin47analogs include E. coli, Bacillus species, Haemophilus Bordetella fungi,yeast, mammalian cells or the baculovirus expression system may be used.

Thus, in accordance with the invention, it may be preferred to make theHin47 analog protein by recombinant methods. Particularly desirablehosts for expression in this regard include Gram positive bacteria whichdo not have LPS and are therefore endotoxin free. Such hosts includespecies of Bacillus and may be particularly useful for the production ofnon-pyrogenic Hin47 analog.

Biological Deposits

Plasmid DS-1011-1-1 pT7/Hin47* that contains a portion coding for aHin47 analog that is described and referred to herein has been depositedwith the American Type Culture Collection (ATCC) located at Rockville,Maryland, USA, pursuant to the Budapest Treaty on Jul. 27, 1994 underATCC American No. 75845. Samples of the deposited plasmid will becomeavailable to the public upon grant of a patent based upon this UnitedStates patent application. The invention described and claimed herein isnot to be limited in scope by plasmid deposited, since the depositedembodiment is intended only as an illustration of the invention. Anyequivalent or similar plasmids that encode similar or equivalentantigens as described in this application are within the scope of theinvention.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example illustrates the cloning of the hin47 gene from non-typableH. influenzae strain SB33.

Chromosomal DNA was prepared from H. influenzae strain SB33, and anEMBL3 library was prepared and screened with a labelled oligonucleotideprobe specific for the 5'-end of hin47. Non-typable H. influenzae strainSB33 was grown on Mueller-Hinton agar or in brain heart infusion brothas described by Harkness et al., 1992. Chromosomal DNA was prepared asfollows: cells from 50 ml of culture were pelleted by centrifugation at5000 rpm for 15 to 20 min, at 4° C., in a Sorvall RC-3B centrifuge. Thecell pellet was resuspended in 10 ml of TE (10 mM Tris/HCl, 1 mM EDTA,pH 7.5), pronase was added to 500 μg ml⁻¹ and SDS to 1%. The sample wasincubated at 37° C. until a clear lysate was obtained. The lysate wasgently extracted once with Tris-saturated phenol (pH 7.4), once withTris-saturated phenol/chloroform (1:1) and once with chloroform. Thefinal aqueous phase was dialysed at 4° C. for 24 h against 1M NaCl,followed by 24 h against TE.

An EMBL3 library was prepared by partial digestion of SB33 chromosomalDNA with Sau3A I, followed by size fractionation either on a 10 to 30%sucrose gradient in TNE (20 mM Tris/HCl, 5 mM NaCl, 1 mM EDTA, pH 8.0)or by preparative gel electrophoresis. Fractions containing DNAfragments greater than 5 kb in length were pooled, precipitated andligated with BamH I arms of EMBL3 (Promega). The ligation mixture waspackaged using a Gigapack II packaging kit and plated onto E. coli LE392cells. The libraries were amplified and stored at 4° C. in the presenceof 0.3% chloroform.

Plaques were lifted onto nitrocellulose filters for hybridization with a³² p-labelled oligonucleotide probe (3026.SL). The oligonucleotidesequence was ATGAAAAAAACACGTTTTGTATTAAATAGTATTGCACTTGG (SEQ ID NO: 3)corresponding to the N-terminal amino acid sequence MKKTRFVLNSIALG (SEQID NO: 19). Phage DNA was prepared from putative plaques and the insertDNA was excised by Sal I digestion and cloned into pUC8-BgXb digestedwith Sal I. Plasmids JB-1031-1-14 and JB-1068-2-2 (FIG. 1) were selectedfor further analysis.

Example 2

This Example illustrates the characterization and sequence analysis ofthe hin47 gene and deduced amino acid sequence of the Hin47 protein fromNTHi strain SB33.

Restriction mapping and Southern blot analysis of clones JB-1031-1-14and JB-1068-2-2 localized the hin47 gene on a 4.7 kb BamH I/BamH I or a2.7 kb BamH I/Pst I DNA fragment. The 4.7 kb BamH i/BamH I fragment fromJB-1068-2-2 was subcloned into pUC8/BgXb generating plasmid DS-755-1.The 3.1 kb BamH I to Xba I fragment of DS-755-1 was subcloned into pUC18generating plasmid JB-1165-1 which has restriction sites suitable forthe Erase-a-base (Promega) procedure (FIG. 1). This technique generatessuccessive clones with increasing truncations of insert DNA, with thedeletions occurring from the same end. The resultant nested set ofclones can be sequenced rapidly using a universal primer.

DNA from plasmid JB-1165-1 was digested with BamH I and Sac I andsubjected to exoIII digestion using an Erase-a-base kit. The resultantset of truncated plasmids was analysed by agarose gel electrophoresisand representative plasmids were selected for sequence analysis.

Plasmid DNA for sequencing was prepared by a modification of theprocedure of Holmes and Quigley, 1981. Briefly, the cell pellet from 50ml of culture was resuspended in 10 ml STET (8% sucrose, 5% TritonX-100, 50 mM EDTA, and 50 mM Tris/HCl, pH 8.0), lysozyme (2.5 mg) wasadded and the mixture was boiled for 2 min. The sample was spun at14,000 rpm in a Sorvall RC 5B for 20 minutes and the supernatant wasprecipitated with an equal volume of isopropanol, washed with 70%ethanol then absolute ethanol, and then air dried. The pellet wasresuspended in 0.9 ml of TE, then 20 μl of 5 mg ml⁻¹ RNAse A were added,and the mixture was incubated at 37° C. for 15 min. After the additionof 500 μl of 1.5M NaCl/30% PEG, the mixture was incubated on ice for 30min and the DNA was pelleted by centrifugation in an Eppendorf microfugefor 10 min. The pellet was resuspended in 400 ∥l of TE and extractedtwice with Tris-saturated phenol (pH 7.4), twice with Tris-saturatedphenol/chloroform (1:1) and twice with chloroform. The DNA wasprecipitated by adding 40 μl of 3M ammonium acetate and 1 ml of ethanol,washed with 70% ethanol and resuspended in distilled water.

DNA samples were sequenced using the ABI model 370A DNA sequencer andthe dye terminator chemistry. The universal reverse primer was used withthe nested set of clones to determine the sequence of the hin47 codingstrand. Oligonucleotide primers of approximately 25 bases in length wereused to confirm the sequence of the non-coding strand. The nucleotidesequence of the SB33 hin47 gene and the deduced amino acid sequence ofthe Hin47 protein are shown in FIG. 2. The nucleotide and N-terminalamino acid sequences of Hin47 presented at the ASM meeting, New Orleans,May 26 to 30, 1992 are indicated in lower case in FIG. 2. The aminoterminal sequences of the SB33 Hin47 and this presented sequence areidentical, establishing the identity of the cloned gene as hin47.

Example 3

This Example describes the discovery of serine protease activity ofHin47 protein.

The deduced amino acid sequence of Hin47 protein determined in Example 2above was compared with all other known proteins in the Genbank database. As described above, Hin47 protein is described in published PCTapplications WO 94/00149, WO 92/11367 and WO 92/10936 to be an adhesinmolecule of Haemophilus. It was, therefore, a surprising and unexpecteddiscovery of the present invention that Hin47 protein has significantamino acid homolgy (55%) with the serine protease E. coli htrA and S.typhimurium htrA and other proteases. These amino acid sequencehomologies are shown in FIGS. 3 and 4. Furthermore, Hin47 protein wasfound to autodigest unless it was stored in the presence of a serineprotease inhibitor, such as Pefablock.

Example 4

This Example illustrates the generation of the mutant hin47 gene bysite-directed mutagenesis.

As explained above, H. influenzae Hin 47, E. coli htrA, and S.typhimurium htrA are all serine proteases. The consensus sequence of theactive site of serine proteases is GDSGGPK (SEQ ID NO: 18) [Brenner,1988] with serine being the active residue. The htrA proteins both havea GNSGGAL (SEQ ID NO: 17) sequence and in H. influenzae Hin47, there isthe identical sequence between residues 195 and 201 of the matureprotein. Thus, the serine residue at position 197 was selected forsite-directed mutagenesis, to produce an analog of Hin47 with reducedprotease activity.

An oligonucleotide CGCTCCACCAGCATTACCGCGG (SEQ ID NO: 20) wassynthesized which would change the serine residue at 197 to an alanine.The hin47 gene was cloned into M13mp18 generating clone DS-981-3 andmutagenesis was performed using the Amersham In Vitro Site-DirectedMutagenesis kit. Clone DS-991-8 was confirmed by sequence analysis tocontain the mutation Serine-197 to Alanine. This mutant hin47 gene isdesignated hin47*.

In addition a comparison of the amino acid sequence of Hin47 with otherproteases (as shown in FIG. 4) revealed that amino acids His-91 andAsp-121 are sites appropriate for mutagenesis to produce an analog ofHin47 with reduced protease activity. By mutagenesis methods analogousto those described above, His-91 and/or Asp-121 are deleted or replacedby different amino acids. Such amino acid replacements may includeHis-91 to Alanine and Asp-121 to Alanine. Olignonucleotides to effectsuch mutagenesis include:

His-91→Ala-91 5' ATCAATAACAGCATTATTGGT 3' (SEQ ID NO: 21)

Asp-121→Ala-121 5' TAATGCAATTGCTGATAGTTC3' (SEQ ID NO: 22)

Many serine proteases are secreted in an inactive (`zymogen`) form, andrequire clipping to expose their active sites. N terminal sequenceanalysis of mature natural Hin47 protein suggested the cleavage of thepreprotein to occur at KFFG DRFAEQ (SEQ ID NO: 23). Modifications ofamino acids that prevent cleavage of the molecule to produce the activeprotease molecule can produce an analog of Hin47 having reduced proteaseactivity.

Example 5

This Example illustrates the construction of plasmids expressing Hin47Ser-197→alanine analog from E. coli.

The mutated hin47* gene from plasmid DS-991-8 was cloned into the pT7-7expression vector to generate plasmid DS-1011-1-1 (FIG. 5). E. colistrain BL21/DE3 was transformed to generate E. coli strain DS-1018-3-1which expresses Hin47 Ser197→alanine analog upon induction.

In order to utilize tetracycline selection, the hin47* gene was clonedinto pBR328. The Bgl II/Cla I T7/hin47* gene fragment from DS-1011-1-1was cloned into pEVvrf1 (Young and Davis, 1985) in order to generate aBgl II/BamH I fragment which could be cloned into pUC-4K (Pharmacia)digested with BamH I. The resultant clone DS-1034-3 was digested withEcoR I and the T7/hin47* gene fragment cloned into pBR328 (BoehringerMannheim Corporation) to generate plasmids DS-1048-2 and DS-1067-2.Electroporation of plasmid DNA into E. coli strain BL21DE3 resulted instrains DS-1071-1-1 and DS-1071-3-1 which express the Hin47Ser-197→alanine analog.

Example 6

This Example illustrates the expression of Hin47 Ser-197→alanine analogfrom E. coli.

An overnight culture of strains DS-1018-3-1, DS-1071-1-1, or DS-1071-3-1were grown overnight in NZCYM media+3% dextrose+antibiotics (ampicillinat 25 μg ml⁻¹ or tetracycline at 10 μg ml⁻¹), at 37° C., with shaking. A1:40 dilution of the overnight culture was inoculated into the samemedium and grown at 37° C. with shaking until the absorbance was A₅₇₈approximately 0.3. A 1/10 volume of 10% lactose was then added to induceexpression from the T7 promoter. Cell samples were harvested about 4hours after induction by centrifuging culture samples at 5000 rpm for 10min in a Sorvall RC-3B, at 4° C.

Example 7

This Example illustrates the extraction and purification of Hin47.

Hin47 was expressed as soluble protein in E. coli. The cell pellet froma 250 ml culture, prepared as described in Example 6, was resuspended in40 ml of 50 mM Tris-HCl, pH 8.0, and disrupted by sonication (3×10 min,70% duty circle). The extract was centrifuged at 20,000×g and theresulting supernatant which contained >95% of the soluble Hin47 proteinwas retained. This fraction was called "Hin47-extract".

This Hin47-extract was further purified on a DEAE Sephacel column. Fortyml of the Hin47-extract was applied onto a 20-ml DEAE Sephacel columnequilibrated in 50 mM Tris-HCl, pH 8.0. Hin47 bound to the column underthese conditions. The column was washed with 100 ml of 50 mM Tris-HCl,pH 8.0, and then washed with 100 ml of 500 mM Tris-HCl, pH 8.0containing 20 mM NaCl. Hin47 was then eluted with 50 mM Tris-HCl, pH8.0, containing 40 mM NaCl. The amount of Hin47 in the fractions wasdetermined by the BCA protein assay. The purity of Hin47 was assessed bySDS-PAGE analysis. The fractions containing Hin47 were combined andstored at -20° C.

Example 8

This Example illustrates the extraction and purification of Hin47Ser-197→alanine analog.

Hin47 Ser-197→alanine analog was expressed in inclusion bodies in E.coli. The cell pellet from a 250 ml culture, prepared as described inExample 6, was resuspended in 40 ml of 50 mM Tris-HCl, pH 8.0, anddisrupted by sonication (3×10 min, 70% duty circle). The extract wascentrifuged at 20,000×g and the resulting pellet was saved. The pelletwas re-extracted with 40 ml of 50 mM Tris-HCl, 0.5% Triton X-100, 10 mMEDTA, pH 8.0. The suspension was sonicated 10 min at 70% duty circle.The extract was centrifuged at 300×g for 5 min. The resultantsupernatant was centrifuged again at 20,000×g for 30 min and theresultant pellet was saved. The pellet was resuspended in 50 mMTris-HCl, 0.5% Triton X-100, 10 mM EDTA, pH 8.0. The suspension was thenmixed with 50 mM Tris-HCl, pH 8.0 containing 8M urea. The final ureaconcentration in the mixture was adjusted to 2M with 50 mM Tris-HCl, pH8.0. Hin47 Ser-197→alanine analog was completely solubilized under thereconditions. The final volume of the solution was 20 ml. This fraction iscalled "Hin47 analog-extract". The Hin47 analog-extract was furtherpurified on a DEAE Sephacel column. Twenty ml of Hin47 analog-extractwas applied onto a 10 ml DEAE Sephacel column equilibrated in 50 mMTris-HCl, pH 8.0. Hin47 Ser-197→alanine analog bound to the column underthese conditions. The column was washed with 50 mM Tris-HCl, pH8.0, andHin47 analog was eluted with 50 mM Tris-HCl, pH 8.0, containing 30 mMNaCl. The amount of Hin47 analog in the fractions was determined by theBCA protein assay. The purity of Hin47 analog was assessed by SDS-PAGEanalysis (FIG. 6). The fractions containing Hin47 analog were combinedand stored at -20° C.

Example 9

This Example illustrates the protease activity of Hin47 and Hin47Ser-197→alanine analog.

The enzymatic activity of Hin47 and Hin47 Ser-197→alanine analog wasanalyzed using β-casein as a substrate (FIG. 7). The reaction mixturescontained 5 μg of β-casein and either Hin47 or Hin47 analog. Thereaction was carried out at 37° C. for two hours, and then stopped byadding the SDS-sample buffer and instantly heating the sample at 100° C.for 5 min. The aliquots were anlyzed by SDS-PAGE. As shown in FIG. 7,digestion of β-casein by Hin47 was more obvious after two hours (panelA, lane 1) in comparison to the fractions containing Hin47 analog (panelA, lane 2) or without any exogenous proteins (panel A, lane 3). Thepresence of Hin47 or Hin47 analog in these mixtures were confirmed byimmuno-blotting using a monoclonal antibody to Hin47 (FIG. 7, panel C,lanes 1 and 2).

The protease activities of Hin47 and Hin47 Ser-197→alanine analog werealso compared by analyzing the autodigestion of Hin47 or Hin47 analog at4° C. and -20° C. The purified Hin47 or analog were stored at either 4°C. or -20° C. for up to 20 days. Aliquots were taken on days 0, 10 and20 and the stability of Hin47 or analog was analyzed by immuno-blottingusing a Hin47 monoclonal antibody (FIG. 8). The analog was much morestable than Hin47 up to 20 days when stored at either 4° C. or -20° C.

To further examine the protease activity of the Hin47 Ser-197→alanineanalog, the ability of Hin47 or analog to degrade an 80kDa H. influenzaerecombinant antigen was examined. Thus, a mixed antigen study wasperformed to determine the proteolytic effect of Hin47 or Hin 47 analogon another antigen. An 80 kDa H. influenzae recombinant protein (TBP1)was chosen for this study in order to distinguish it from the Hin47 oranalog protein (47 kDa). Five mixtures were formulated as follows:80-kDa). Five mixtures were formulated as follows: 80-kDa protein alone;80-kDa protein+Hin47; 80-kDa protein+analog; Hin47 alone; and analogalone. The amount of each protein in the mixture was 5 μg. The mixtureswere stored at 4° C. up to four weeks. Aliquots were taken on days 0,7,14 and 28 for analysis by SDS-PAGE (FIG. 9). Both the 80 kDa proteinand Hin47 were largely degraded after one week (lanes 2 and 4). Incontrast, the 80 kDa protein in combination with Hin47 analog remainedintact after one week, and showed only slight degradation even afterfour weeks (lane 3).

Example 10

This Example illustrates the comparative immunogenicity of Hin47 andHin47 analog in mice. The results of a study to determine thecomparative immunogenicity of Hin47 and the Hin47 Ser-197→alanine analogare shown in FIG. 10. Thus, groups of five Balb/c mice were injectedthree times (as indicated by arrows) s.c. on days 1, 29 and 43 with 1-μgdose of either Hin47 or Hin47 analog in the presence of AlPO₄ (1.5 mgper dose). Blood samples were taken on days 14, 28, 42 and 56 (asindicated by bleeds 1, 2, 3 and 4, respectively) for analyzing theanti-Hin47 antibody titers by EIAs. The determination of anti-Hin47antibodies in mouse sera was performed as described by Panezutti et al.(1993). Microtiter wells were coated with 1 μg of either Hin47 or analogfor 16 hours at room temperature. The plates were then blocked with 0.1%(w/v) bovine serum albumin in PBS. The mouse sera were serially diluted,added to the wells, then incubated for one hour at room temperature.Affinity-purified F(ab')₂ fragments of goat anti-mouse IgG (Fc specific)antibody conjugated to horseradish peroxidase were used as the secondantibody. The reactions were developed using tetramethylbenzidine (TMB/H₂ O₂) and absorbencies were measured at 450 nm (using 540 nm as areference wavelength) in a Flow Multiskan MCC microplate reader. Thereactive titer of an antiserum was defined as the reciprocal of thedilution consistently showing a two-fold increase in absorbance overthat obtained with the pre-bleed serum sample. As can be seen from FIG.10, both Hin47 and the Hin47 analog elicited comparable IgG titers inmice regardless of whether Hin47 or mutant was used as an anitgen inEIAs.

To further examine the immune response to Hin47 or the Hin47Ser-197→alanine analog, the subclasses of anti-Hin47 IgG in mouse serawere determined. Microtiter wells sere coated with 1 μg of purifiedHin47 or analog. The final bleed of mouse serum samples from thecomparative immunogenicity study (as described above) were pooled andtested in EIAs. Rat anti-mouse IgG₁, IgG_(2a), IgG_(2b) antibodiesconjugated horseradish peroxidase and rabbit anti-mouse IgG₃ conjugatedto horseradish peroxidase were used as reagents in EIAs. The workingdilution of each conjugate was determined using purified antibodysubclasses to avoid cross reactivity. The reactive titers weredetermined as described above. As shown in Table 1 below, theIgG-subclass profile induced in mice by either Hin47 or Hin47 analogwere identical, regardless of whether Hin47 or analog was used as asolid antigen in the EIAs. The predominant IgG response in both groupsof mouse sera was of the IgG₁ isotype. Hence, the Hin47 analog exhibitedsubstantially the same immunogenic properties as the natural protein.

Example 11

This example illustrates the immunoprotective properties of Hin47 andHin47 Ser-197→alanine analog.

The immunoprotective properties of Hin47 and the Hin47 Ser-197→alanineanalog were analyzed by the ability of Hin47 specific antisera toprotect infant rats against H. influenzae type b strain MinnA in abacteremia model. The results of this study are shown in Table 2 below.Groups of nine 6-day old Wistar infant rats were inoculatedsubcutaneously (s.c.) on the dorsum close to the neck with 0.1 mL ofeither a rabbit anti-Hin47 analog antiserum or the correspondingprebleed serum. Twenty-four hours later, the animals were challengedintraperitoneally (i.p.) with 700 cfu of freshly grown Hib strain MinnA.Blood samples were collected 20 hours post-challenge and plated ontochocolate agar plates. Bacterial colonies were counted after 24 hours.As shown in Table 2, three out of nine animals in the group inoculatedwith anti-Hin47 analog antiserum did not show any bacteremia in theirblood. Only one mouse in the group inoculated with anti-Hin47 analogantiserum (11%) had a higher bacteria recovery from the blood samplecompared to mice inoculated with prebleed serum. In contrast, bacteriawere recovered from all the nine mice inoculated with pre-bleed serum.Four out of nine animals (44%) in the group inoculated with pre-bleedserum showed high levels (500 to 1,000) of bacteria recovered in bloodsamples.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novelanalog of Haemophilus influenzae Hin47 protein which has a decreasedprotease activity of less than about 10% of that of the natural Hin47protein as well as isolated and purified DNA molecules encoding thesame.

                  TABLE 1                                                         ______________________________________                                        Hin47 IgG titers in mouse immune sera                                                IgG titers in Group #1*                                                                     IgG titers in Group #2*                                  IgG Suclass                                                                            To Hin47  To Mutant To Hin47                                                                             To Mutant                                 ______________________________________                                        IgG(H + L)                                                                             102,400   102,400   102,400                                                                              102,400                                   IgG.sub.1                                                                               25,600    25,600    25,600                                                                               25,600                                   IgG.sub.2a                                                                             <100      <100      <100   <100                                      IgG.sub.2b                                                                              400       400       400    400                                      IgG.sub.3                                                                              <100      <100      <100   <100                                      ______________________________________                                         *Group #1: Immune sera were pooled from a group of five mice received         Hin47 immunization.                                                           Group #2: Immune sera were pooled from a group of five mice received Hin4     mutant immunization.                                                          Plates were coated with either Hin47 or mutant protein.                  

                  TABLE 2                                                         ______________________________________                                        Protective ability of rabbit Anti-Hin47 Mutant                                antiserum against Hib in infant rat model of bacteremia                              Number of Animals                                                             cfu of Bacteria/2.5 μL Blood                                                        Av.50    Av.200 Av.650  Total                                 Antibody Av.0   (10-100) (100-300)                                                                            (300-1,000)                                                                           Animals                               ______________________________________                                        Anti-Hin47*                                                                            3      3        2      1       9                                     Prebleed 0      4        1      4       9                                     ______________________________________                                    

Groups of nine 6-day old infant rats were immunized s.c. with either arabbit anti-Hin47 mutant antiserum or the corresponding prebleed serum.Animals were challenged i.p. with 700 cfu H. influenzae strain MinnAafter 24 hours. The blood samples were taken at 20 hours after thechallenge.

Anti-47* antibody: rabbit immune serum raised against purified Hin47mutant in CFA/IFA.

Average bacteria recovery from immunized group: 100 cfu per 2.5 μL ofblood; from control group: 290 cfu per 2.5 μL of blood.

Reference List

1. Zangwill et al, 1993 MMWR 42:1-15.

2. Schoendorf et al, 1994 Pediatrics 93:663-8.

3. Brenner et al, 1988 Nature 334:528-530.

4. O'Hagan 1992 Clin. Pharmokinet. 22:1-10.

5. Ulmer et al, 1993 Curr. Opinion. Invest. Drugs 2:983-989.

6. Chang et al, 1978 Nature 275:617.

7. Goeddel et al 1980 Nucl. Acid. Res. 8:4057.

8. Harkness et al, 1992 J. Bacteriol. 174:2425-2430.

9. Loeb et al, 1987 Infec. immun. 55:2612-2618.

10. Holmes and Quigley 1981. Analyt. Biochem. 114:193-197.

11. Young and Davis 1985 Gene 38:31-38.

12. Panezutti et al, 1993 Infec. Immun. 61:1867-72.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 23                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2894 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATCCGTTAA TACTGAAATAAATGGCACACCTTTTTCACGCATTTGGGCAAGTACAGCA60               CTGGTTTTTGCCATTTGCATTAAAGAGAATAATGCTTCCTGCATACGAGCACCACCACTC120               GCAGAGAAACATACAAACGGACAATTCATTTCCATCGCTTTTTCAGCCGCTTTAA CAAAT180              TTTGCACCAACTACAGAACCCATTGAACCGCCCATAAAAGCAAAGTTCGATGCAGCCACA240               ACAATTGGCATATCATAAAGTGTACCTGTCATAGTAATTAGCGCATCTTTCTCGCCCGTT300               TCTTTTTGTGCCGCATTGATACGATCTTTA TATTTCTTTAAATCTTTAAATTTTAAAATA360              TCTTTTGGTTCTAAATCTGCCGCAATTTCTTGGCTTGAATCTTCGTCCAATAAATTTAAT420               AAACGCTCACGAGCATCAATACGCATATGATGACCACATTTCGGGCAAACATACAGATTA480               CGTT TGAGTTCTTCACTATAAAGTACTTGTTCACAAGCAGTACATTTTGTCCATACGCCT540              TCTGGCACATTGGCTTTTCGAGTGGAAGAAGAAGGACTTTTACTAAAAATTCGGTTAATC600               CAGCTCATTTTTTGACCTTTTTATTGACTAGAAAATTGCGCGTATTAG AACATAAATTTA660              TAGAATTTGCTACTTGTAAGACCGTTTTTGTACTGCTCCGATTTCCTTTTAAACAAGATA720               ATTTGCTCTCCTCTTATTGAACATTTTTTTTATTTTTTTGTCTTACTGACCACGTTATCT780               GAAATTTATTTTGGAGTATTTA TGAAAAAAACACGTTTTGTACTAAATAGTATTGCACTT840              GGATTAAGTGTATTAAGCACATCATTTGTTGCTCAAGCCACTTTGCCAAGTTTTGTTTCG900               GAACAAAACAGTCTTGCACCAATGTTAGAAAAAGTACAACCTGCCGTTGTCACTCTTTCC960               GTTGAAGGAAAAGCTAAAGTAGATTCTCGTTCTCCTTTCCTAGACGATATTCCTGAAGAA1020              TTTAAATTCTTCTTTGGCGATCGTTTTGCCGAACAATTTGGTGGACGTGGAGAATCAAAG1080              CGTAACTTCCGTGGTTTAGGTTCTGGTGTCATTATTAATG CAAGCAAAGGCTATGTTTTA1140             ACCAATAATCATGTTATTGATGAAGCTGATAAAATTACCGTGCAATTACAAGATGGGCGT1200              GAATTTAAAGCAAAATTAGTGGGTAAAGATGAACTATCAGATATTGCATTAGTACAGCTT1260              GAAAAACCAAGTAAT TTAACAGAAATCAAATTTGCTGATTCCGACAAATTACGCGTAGGC1320             GATTTCACTGTTGCAATCGGTAATCCATTTGGTTTAGGTCAAACTGTGACATCAGGTATT1380              GTTTCTGCATTGGGTCGTTCAACAGGTTCTGACAGTGGCACTTATGAAAACTATATTCA A1440             ACCGATGCAGCAGTAAACCGCGGTAATTCGGGTGGAGCGTTAGTAAACTTAAATGGCGAA1500              CTTATTGGAATTAATACCGCAATTATTTCTCCAAGCGGTGGCAATGCAGGAATTGCCTTT1560              GCGATTCCAAGTAATCAAGCAAGCAATTTAGTG CAACAAATTTTAGAATTTGGTCAAGTG1620             CGTCGCGGATTGCTTGGTATTAAAGGTGGCGAACTCAATGCTGATTTAGCCAAAGCCTTT1680              AATGTAAGCGCGCAACAAGGCGCATTTGTAAGTGAAGTTTTACCGAAATCTGCTGCTGAA1740              AAAGCAGG ACTTAAAGCGGGCGATATTATCACGGCGATGAACGGTCAAAAAATCTCAAGT1800             TTCGCTGAAATTCGTGCAAAAATCGCAACCACTGGTGCAGGCAAAGAGATTAGCTTGACT1860              TACTTACGTGATGGCAAATCCCACGACGTTAAAATGAAATTACAAGCGGAT GATAGTAGC1920             CAACTTTCCTCAAAAACTGAGTTGCCTGCATTAGATGGTGCAACATTGAAAGACTACGAT1980              GCTAAAGGCGTTAAAGGAATTGAAATCACAAAAATTCAACCTAATTCGCTGGCTGCACAA2040              CGTGGTTTAAAATCGGGCGATATTAT TATTGGTATTAATCGTCAAATGATCGAAAACATT2100             CGTGAATTAAATAAAGTGCTTGAAACTGAACCGTCAGCAGTTGCACTTAATATTTTACGA2160              GGTGACAGTAATTTCTATTTATTAGTGCAATAATCTGCTTGATATATTTAAGAAAAAAGT2220               CCGATCACAATGATCGGGCTTCTTTTTATGCAGCAATCGTTCTTAACAAATCCACCACAA2280             ATTCTAACCGCACTTTGTTATCAGATAAATCTTTCATGAACTTAAATTTTAATGGGCCAT2340              CAAATCGATACACAATAGGTTCTTTTTGAATTAATTGAATAAAT TTATCTGGATTCACTT2400             GTGCTTTTGCTGAAAACTCAATAAAACCGCCTTGTGTTCCTGCATCAATTCGCACAACTT2460              TCAACGGCTCAACCAACAAACGCAATTCTGCAATTTGCAGTAAATTTTTTGTTGCATCAG2520              GCAATAATCCGAATCGATC TATTAACTCAACTTTTAATTCATCTAATTCTGCTTTACTCT2580             CTGCTGCAGCAATGCGTTTATAAAAGGATAAACGCATATTCACGTCTCCTAGATAATCAT2640              CAGGCAGTAAAGCAGGCACACGCAATTCAATATCCGCTTGTTGTTGCGTCAATTCTTCTA 2700             ATGATGGTTCACGCCCTTCTTTTAACGCTTTAACCGCTGCATCCAATAATTCCATATAAA2760              GCGAAAAACCGATGCTTTCAATTTGTCCACTTTGTTCGTTTCCAAGTAATTCGCCGGCAC2820              CACGAATCTCTAAATCGTGGGTTGCCAAGATAAAACC AGCCCCAAGATTATCAAGATTTT2880             CCAAGGCATCTAGA2894                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 463 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysLysThrArgPheValLeuAsnSerIleAlaLeuGlyLeuSer                              151015                                                                        ValLeuSerThrSerPheVa lAlaGlnAlaThrLeuProSerPheVal                             202530                                                                        SerGluGlnAsnSerLeuAlaProMetLeuGluLysValGlnProAla                              35 4045                                                                       ValValThrLeuSerValGluGlyLysAlaLysValAspSerArgSer                              505560                                                                        ProPheLeuAspAspIleProGluGlu PheLysPhePhePheGlyAsp                             65707580                                                                      ArgPheAlaGluGlnPheGlyGlyArgGlyGluSerLysArgAsnPhe                              85 9095                                                                       ArgGlyLeuGlySerGlyValIleIleAsnAlaSerLysGlyTyrVal                              100105110                                                                     LeuThrAsnAsnHisV alIleAspGluAlaAspLysIleThrValGln                             115120125                                                                     LeuGlnAspGlyArgGluPheLysAlaLysLeuValGlyLysAspGlu                              130 135140                                                                    LeuSerAspIleAlaLeuValGlnLeuGluLysProSerAsnLeuThr                              145150155160                                                                  GluIleLysPheAla AspSerAspLysLeuArgValGlyAspPheThr                             165170175                                                                     ValAlaIleGlyAsnProPheGlyLeuGlyGlnThrValThrSerGly                               180185190                                                                    IleValSerAlaLeuGlyArgSerThrGlySerAspSerGlyThrTyr                              195200205                                                                     GluAsnTyrIle GlnThrAspAlaAlaValAsnArgGlyAsnSerGly                             210215220                                                                     GlyAlaLeuValAsnLeuAsnGlyGluLeuIleGlyIleAsnThrAla                              225 230235240                                                                 IleIleSerProSerGlyGlyAsnAlaGlyIleAlaPheAlaIlePro                              245250255                                                                     SerAs nGlnAlaSerAsnLeuValGlnGlnIleLeuGluPheGlyGln                             260265270                                                                     ValArgArgGlyLeuLeuGlyIleLysGlyGlyGluLeuAsnAlaAsp                               275280285                                                                    LeuAlaLysAlaPheAsnValSerAlaGlnGlnGlyAlaPheValSer                              290295300                                                                     GluValLeuP roLysSerAlaAlaGluLysAlaGlyLeuLysAlaGly                             305310315320                                                                  AspIleIleThrAlaMetAsnGlyGlnLysIleSerSerPheAlaGlu                               325330335                                                                    IleArgAlaLysIleAlaThrThrGlyAlaGlyLysGluIleSerLeu                              340345350                                                                      ThrTyrLeuArgAspGlyLysSerHisAspValLysMetLysLeuGln                             355360365                                                                     AlaAspAspSerSerGlnLeuSerSerLysThrGluLeuProAlaLeu                               370375380                                                                    AspGlyAlaThrLeuLysAspTyrAspAlaLysGlyValLysGlyIle                              385390395400                                                                   GluIleThrLysIleGlnProAsnSerLeuAlaAlaGlnArgGlyLeu                             405410415                                                                     LysSerGlyAspIleIleIleGlyIleAsnArgGlnMetIle GluAsn                             420425430                                                                     IleArgGluLeuAsnLysValLeuGluThrGluProSerAlaValAla                              43544044 5                                                                    LeuAsnIleLeuArgGlyAspSerAsnPheTyrLeuLeuValGln                                 450455460                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGAAAAAAACACGTTTTGTATTAAATAGTATTGCACTTGG41                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C ) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLysLysThrArgPheValLeuAsnSerIleAlaLeuGlyLeuSer                              151015                                                                        ValLeuSerThr SerPheValAlaGlnAlaThrLeuProSerPheVal                             202530                                                                        SerGluGlnAsnSer                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 472 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetLysLysThrThrLeuAlaLeuSerArgLeuAlaLeuSerLeuSer                              1510 15                                                                       LeuAlaLeuSerProLeuSerAlaThrAlaAlaGluThrSerSerAla                              202530                                                                        ThrThrAlaGlnGlnMetProSerLeuAlaPro MetLeuGluLysVal                             354045                                                                        MetProSerValValSerIleAsnValGluGlySerThrThrValAsn                              5055 60                                                                       ThrProArgMetProArgAsnPheGlnGlnPhePheGlyAspAspSer                              65707580                                                                      ProPheCysGlnGluGlySerProPheGlnSerSe rProPheCysGln                             859095                                                                        GlyGlyGlnGlyGlyAsnGlyGlyGlyGlnGlnGlnLysPheMetAla                              100105 110                                                                    LeuGlySerGlyValIleIleAspAlaAspLysGlyTyrValValThr                              115120125                                                                     AsnAsnHisValValAspAsnAlaThrValIl eLysValGlnLeuSer                             130135140                                                                     AspGlyArgLysPheAspAlaLysMetValGlyLysAspProArgSer                              145150155 160                                                                 AspIleAlaLeuIleGlnIleGlnAsnProLysAsnLeuThrAlaIle                              165170175                                                                     LysMetAlaAspSerAspAlaLeuA rgValGlyAspTyrThrValGly                             180185190                                                                     IleGlyAsnProPheGlyLeuGlyGluThrValThrSerGlyIleVal                              195 200205                                                                    SerAlaLeuGlyArgSerGlyLeuAsnAlaGluAsnTyrGluAsnPhe                              210215220                                                                     IleGlnThrAspAlaAlaIleAsnArgGly AsnSerGlyGlyAlaLeu                             225230235240                                                                  ValAsnLeuAsnGlyGluLeuIleGlyIleAsnThrAlaIleLeuAla                              245 250255                                                                    ProAspGlyGlyAsnIleGlyIleGlyPheAlaIleProSerAsnMet                              260265270                                                                     ValLysAsnLeuThrSer GlnMetValGluTyrGlyGlnValLysArg                             275280285                                                                     GlyGluLeuGlyIleMetGlyThrGluLeuAsnSerGluLeuAlaLys                              290 295300                                                                    AlaMetLysValAspAlaGlnArgGlyAlaPheValSerGlnValLeu                              305310315320                                                                  ProAsnSerSerAlaAl aLysAlaGlyIleLysAlaGlyAspValIle                             325330335                                                                     ThrSerLeuAsnGlyLysProIleSerSerPheAlaAlaLeuArgAla                               340345350                                                                    GlnValGlyThrMetProValGlySerLysLeuThrLeuGlyLeuLeu                              355360365                                                                     ArgAspGlyLysG lnValAsnValAsnLeuGluLeuGlnGlnSerSer                             370375380                                                                     GlnAsnGlnValAspSerSerSerIlePheAsnGlyIleGluGlyAla                              385 390395400                                                                 GluMetSerAsnLysGlyLysAspGlnGlyValValValAsnAsnVal                              405410415                                                                     LysThr GlyThrProAlaAlaGlnIleGlyLeuLysLysGlyAspVal                             420425430                                                                     IleIleGlyAlaAsnGlnIleAlaValLysAsnIleAlaGluIleArg                               435440445                                                                    LysValLeuAspSerLysProSerValLeuAlaLeuAsnIleGlnArg                              450455460                                                                     GlyAspArgHis LeuProValAsn                                                     465470                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 475 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetLysLysThrThrLeuA laMetSerAlaLeuAlaLeuSerLeuGly                             151015                                                                        LeuAlaLeuSerProLeuSerAlaThrAlaAlaGluThrSerSerSer                              20 2530                                                                       AlaMetThrAlaGlnGlnMetProSerLeuAlaProMetLeuGluLys                              354045                                                                        ValMetProSerValVal SerIleAsnValGluGlySerThrThrVal                             505560                                                                        AsnThrProArgMetProArgAsnPheGlnGlnPhePheGlyAspAsp                              6570 7580                                                                     SerProPheCysGlnAspGlySerProPheGlnAsnSerProPheCys                              859095                                                                        GlnGlyGlyGlyA snGlyGlyAsnGlyGlyGlnGlnGlnLysPheMet                             100105110                                                                     AlaLeuGlySerGlyValIleIleAspAlaAspLysGlyTyrValVal                              115 120125                                                                    ThrAsnAsnHisValValAspAsnAlaSerValIleLysValGlnLeu                              130135140                                                                     SerAspGlyArgLysPhe AspAlaLysValValGlyLysAspProArg                             145150155160                                                                  SerAspIleAlaLeuIleGlnIleGlnAsnProLysAsnLeuThrAla                               165170175                                                                    IleLysLeuAlaAspSerAspAlaLeuArgValGlyAspTyrThrVal                              180185190                                                                     AlaIle GlyAsnProPheGlyLeuGlyGluThrValThrSerGlyIle                             195200205                                                                     ValSerAlaLeuGlyArgSerGlyLeuAsnValGluAsnTyrGluAsn                              2 10215220                                                                    PheIleGlnThrAspAlaAlaIleAsnArgGlyAsnSerGlyGlyAla                              225230235240                                                                  LeuVa lAsnLeuAsnGlyGluLeuIleGlyIleAsnThrAlaIleLeu                             245250255                                                                     AlaProAspGlyGlyAsnIleGlyIleGlyPheAlaIleProSerAsn                               260265270                                                                    MetValLysAsnLeuThrSerGlnMetValGluTyrGlyGlnValArg                              275280285                                                                     A rgGlyGluLeuGlyIleMetGlyThrGluLeuAsnSerGluLeuAla                             290295300                                                                     LysAlaMetLysValAspAlaGlnArgGlyAlaPheValSerGlnVal                              305 310315320                                                                 MetProAsnSerSerAlaAlaLysAlaGlyIleLysAlaGlyAspVal                              325330335                                                                     IleThrSerLeuAsnGlyLysProIleSerSerPheAlaAlaLeuArg                              340345350                                                                     AlaGlnValGlyThrMetProValGlySerLysIleSerLeuG lyLeu                             355360365                                                                     LeuArgGluGlyLysAlaIleThrValAsnLeuGluLeuGlnGlnSer                              370375380                                                                      SerGlnSerGlnValAspSerSerThrIlePheSerGlyIleGluGly                             385390395400                                                                  AlaGluMetSerAsnLysGlyGlnAspLysGlyValValVal SerSer                             405410415                                                                     ValLysAlaAsnSerProAlaAlaGlnIleGlyLeuLysLysGlyAsp                              420425 430                                                                    ValIleIleGlyAlaAsnGlnIleProValLysAsnIleAlaGluIle                              435440445                                                                     ArgLysIleLeuAspSerLysProSerValLeuAlaLeu AsnIleGln                             450455460                                                                     ArgGlyAspSerSerIleTyrLeuLeuMetGln                                             465470475                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 228 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       IleValGlyGlyTyrLysCysGluLysAsnSerGlnProTrpGlnVal                              15 1015                                                                       AlaValIleAsnGluTyrLeuCysGlyGlyValLeuIleAspProSer                              202530                                                                        TrpValIleThrAlaAlaHisCy sTyrSerAsnAsnTyrGlnValLeu                             354045                                                                        LeuGlyArgAsnAsnLeuPheLysAspGluProPheAlaGlnArgArg                              5055 60                                                                       LeuValProGlnSerPheArgHisProAspTyrIleProLeuIlePro                              65707580                                                                      ValHisAspHisSerAsnAspLeu MetLeuLeuHisLeuSerGluPro                             859095                                                                        AlaAspIleThrGlyGlyValLysValIleAspLeuProThrLysGlu                              100 105110                                                                    ProLysValGlySerThrCysLeuAlaSerGlyTrpGlySerThrAsn                              115120125                                                                     ProSerGluMetValValSer HisAspLeuGlnCysValAsnIleHis                             130135140                                                                     LeuLeuSerAsnGluLysCysIleGluThrTyrLysAspAsnValThr                              145150 155160                                                                 AspValMetLeuCysAlaGlyGluMetGluGlyGlyLysAspThrCys                              165170175                                                                     AlaGlyAspSerGly GlyProLeuIleCysAspGlyValLeuGlnGly                             180185190                                                                     IleThrSerGlyGlyAlaThrProCysAlaLysProLysThrProAla                              195 200205                                                                    IleTyrAlaLysLeuIleLysPheThrSerTrpIleLysLysValMet                              210215220                                                                     LysGluAsnPro                                                                  225                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 232 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       IleIleGlyGlyArgGluCysGluLysAsnSerHisProTrpGlnVal                              1 51015                                                                       AlaIleTyrHisTyrSerSerPheGlnCysGlyGlyValLeuValAsn                              202530                                                                         ProLysTrpValLeuThrAlaAlaHisCysLysAsnAspAsnTyrGlu                             354045                                                                        ValTrpLeuGlyArgHisAsnLeuPheGluAsnGluAsnThrAlaGln                               505560                                                                       PhePheGlyValThrAlaAspPheProHisProGlyPheAsnLeuSer                              65707580                                                                      Al aAspGlyLysAspTyrSerHisAspLeuMetLeuLeuArgLeuGln                             859095                                                                        SerProAlaLysIleThrAspAlaValLysValLeuGluLeuProThr                              100105110                                                                     GlnGluProGluLeuGlySerThrCysGluAlaSerGlyTrpGlySer                              115120125                                                                      IleGluProGlyProAspAspPheGluPheProAspGluIleGlnCys                             130135140                                                                     ValGlnLeuThrLeuLeuGlnAsnThrPheCysAlaAspAlaHisPro                               145150155160                                                                 AspLysValThrGluSerMetLeuCysAlaGlyTyrLeuProGlyGly                              1651701 75                                                                    LysAspThrCysMetGlyAspSerGlyGlyProLeuIleCysAsnGly                              180185190                                                                     MetTrpGlnGlyIleThrSerTrpGlyHisThrProCysGl ySerAla                             195200205                                                                     AsnLysProSerIleTyrThrLysLeuIlePheTyrLeuAspTrpIle                              210215220                                                                      AspAspThrIleThrGluAsnPro                                                     225230                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 223 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       IleVa lGlyGlyTyrThrCysGlyAlaAsnThrValProTyrGlnVal                             151015                                                                        SerLeuAsnSerGlyTyrHisPheCysGlyGlySerLeuIleAsnSer                               202530                                                                       GlnTrpValValSerAlaAlaHisCysTyrLysSerGlyIleGlnVal                              354045                                                                        Arg LeuGlyGluAspAsnIleAsnValValGluGlyAsnGluGlnPhe                             505560                                                                        IleSerAlaSerLysSerIleValHisProSerTyrAsnSerAsnThr                              65 707580                                                                     LeuAsnAsnAspIleMetLeuIleLysLeuLysSerAlaAlaSerLeu                              859095                                                                         AsnSerArgValAlaSerIleSerLeuProThrSerCysAlaSerAla                             100105110                                                                     GlyThrGlnCysLeuIleSerGlyTrpGlyAsnThrLysSerSerGly                              115120125                                                                     ThrSerTyrProAspValLeuLysCysLeuLysAlaProIleLeuSer                              130135140                                                                     AspS erSerCysLysSerAlaTyrProGlyGlnIleThrSerAsnMet                             145150155160                                                                  PheCysAlaGlyTyrLeuGluGlyGlyLysAspSerCysGlnGlyAs p                             165170175                                                                     SerGlyGlyProValValCysSerGlyLysLeuGlnGlyIleValSer                              180185 190                                                                    TrpGlySerGlyCysAlaGlnLysAsnLysProGlyValTyrThrLys                              195200205                                                                     ValCysAsnTyrValSerTrpIleLysGlnThrIleAlaSerA sn                                210215220                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 228 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      IleValAsnGly GluGluAlaValProGlySerTrpProTrpGlnVal                             151015                                                                        SerLeuGlnAspLysThrGlyPheHisPheCysGlyGlySerLeuIle                               202530                                                                       AsnGluAsnTrpValValThrAlaAlaHisCysGlyValThrThrSer                              354045                                                                        AspValValV alAlaGlyGluPheAspGlnGlySerSerSerGluLys                             505560                                                                        IleGlnLysLeuLysIleAlaLysValPheLysAsnSerLysTyrAsn                              65 707580                                                                     SerLeuThrIleAsnAsnAspIleThrLeuLeuLysLeuSerThrAla                              859095                                                                        AlaSer PheSerGlnThrValSerAlaValCysLeuProSerAlaSer                             100105110                                                                     AspAspPheAlaAlaGlyThrThrCysValThrThrGlyTrpGlyLeu                               115120125                                                                    ThrArgTyrAlaAsnThrProAspArgLeuGlnGlnAlaSerLeuPro                              130135140                                                                     LeuLeuSerAs nThrAsnCysLysLysTyrTrpGlyThrLysIleLys                             145150155160                                                                  AspAlaMetIleCysAlaGlyAlaSerGlyValSerSerCysMetGly                               165170175                                                                    AspSerGlyGlyProLeuValCysLysLysAsnGlyAlaTrpThrLeu                              180185190                                                                      ValGlyIleValSerTrpGlySerSerThrCysSerThrSerThrPro                             195200205                                                                     GlyValTyrAlaArgValThrAlaLeuValAsnTrpValGlnGlnThr                               210215220                                                                    LeuAlaAlaAsn                                                                  225                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 240 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                     ValValGlyGlyThrGluAlaGlnArgAsnSerTrpProSerGlnIle                              151015                                                                        SerLeuGlnTyrArgSerGlySerSerTrpAlaHis ThrCysGlyGly                             202530                                                                        ThrLeuIleArgGlnAsnTrpValMetThrAlaAlaHisCysValAsp                              3540 45                                                                       ArgGluLeuThrPheArgValValValGlyGluHisAsnLeuAsnGln                              505560                                                                        AsnAsnGlyThrGluGlnTyrValGlyValGlnLysIleValV alHis                             65707580                                                                      ProTyrTrpAsnThrAspAspValAlaAlaGlyTyrAspIleAlaLeu                              8590 95                                                                       LeuArgLeuAlaGlnSerValThrLeuAsnSerTyrValGlnLeuGly                              100105110                                                                     ValLeuProArgAlaGlyThrIleLeuAlaAs nAsnSerProCysTyr                             115120125                                                                     IleThrGlyTrpGlyLeuThrArgThrAsnGlyGlnLeuAlaGlnThr                              130135 140                                                                    LeuGlnGlnAlaTyrLeuProThrValAspTyrAlaIleCysSerSer                              145150155160                                                                  SerSerTyrTrpGlySerThrValLysAsnS erMetValCysAlaGly                             165170175                                                                     GlyAspGlyValArgSerGlyCysGlnGlyAspSerGlyGlyProLeu                              180 185190                                                                    HisCysLeuValAsnGlyGlnTyrAlaValHisGlyValThrSerPhe                              195200205                                                                     ValSerArgLeuGlyCysAsnValThr ArgLysProThrValPheThr                             210215220                                                                     ArgValSerAlaTyrIleSerTrpIleAsnAsnValIleAlaSerAsn                              225230 235240                                                                 (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 224 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      IleIleGlyGlyValGluSerIleProHisSerArg ProTyrMetAla                             151015                                                                        HisLeuAspIleValThrGluLysGlyLeuArgValIleCysGlyGly                              2025 30                                                                       PheLeuIleSerArgGlnPheValLeuThrAlaAlaHisCysLysGly                              354045                                                                        ArgGluIleThrValIleLeuGlyAlaHisAspV alArgLysArgGlu                             505560                                                                        SerThrGlnGlnLysIleLysValGluLysGlnIleIleHisGluSer                              657075 80                                                                     TyrAsnSerValProAsnLeuHisAspIleMetLeuLeuLysLeuGlu                              859095                                                                        LysLysValGluLeuThrProAlaValAsn ValValProLeuProSer                             100105110                                                                     ProSerAspPheIleHisProGlyAlaMetCysTrpAlaAlaGlyTrp                              115120 125                                                                    GlyLysThrGlyValArgAspProThrSerTyrThrLeuArgGluVal                              130135140                                                                     GluLeuArgIleMetAspGluLysAlaCysValAs pTyrArgTyrTyr                             145150155160                                                                  GluTyrLysPheGlnValCysValGlySerProThrThrLeuArgAla                              165 170175                                                                    AlaPheMetGlyAspSerGlyGlyProLeuLeuCysAlaGlyValAla                              180185190                                                                     HisGlyIleValSerTyrGlyH isProAspAlaLysProProAlaIle                             195200205                                                                     PheThrArgValSerThrTyrValProTrpIleAsnAlaValIleAsn                              210215 220                                                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 223 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ValValGlyGlyThrArgAlaAlaGlnGlyGluPhePro PheMetVal                             151015                                                                        ArgLeuSerMetGlyCysGlyGlyAlaLeuTyrAlaGlnAspIleVal                              2025 30                                                                       LeuThrAlaAlaHisCysValSerGlySerGlyAsnAsnThrSerIle                              354045                                                                        ThrAlaThrGlyGlyValValAspLeuGlnSerGlyAl aAlaValLys                             505560                                                                        ValArgSerThrLysValLeuGlnAlaProGlyTyrAsnGlyThrGly                              657075 80                                                                     LysAspTrpAlaLeuIleLysLeuAlaGlnProIleAsnGlnProThr                              859095                                                                        LeuLysIleAlaThrThrThrAlaTyrAsnGln GlyThrPheThrVal                             100105110                                                                     AlaGlyTrpGlyAlaAsnArgGluGlyGlySerGlnGlnArgTyrLeu                              115120 125                                                                    LeuLysAlaAsnValProPheValSerAspAlaAlaCysArgSerAla                              130135140                                                                     TyrGlyAsnGluLeuValAlaAsnGluGluIleCysAla GlyTyrPro                             145150155160                                                                  AspThrGlyGlyValAspThrCysGlnGlyAspSerGlyGlyProMet                              1651 70175                                                                    PheArgLysAspAsnAlaAspGluTrpIleGlnValGlyIleValSer                              180185190                                                                     TrpGlyTyrGlyCysAlaArgProGl yTyrProGlyValTyrThrGlu                             195200205                                                                     ValSerThrPheAlaSerAlaIleAlaSerAlaAlaArgThrLeu                                 210215 220                                                                    (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 185 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      IleSerGlyGlyAspAlaIleTyrSerSerThrGlyArgCysSerL eu                             151015                                                                        GlyPheAsnValArgSerGlySerThrTyrTyrPheLeuThrAlaGly                              2025 30                                                                       HisCysThrAspGlyAlaThrThrTrpTrpAlaAsnSerAlaArgThr                              354045                                                                        ThrValLeuGlyThrThrSerGlySerSerPheProAsnAsnAsp Tyr                             505560                                                                        GlyIleValArgTyrThrAsnThrThrIleProLysAspGlyThrVal                              65707580                                                                      GlyGlyGlnAspIleThrSerAlaAlaAsnAlaThrValGlyMetAla                              859095                                                                        ValThrArgArgGlySerThrThrGlyThrHisSerGlyS erValThr                             100105110                                                                     AlaLeuAsnAlaThrValAsnTyrGlyGlyGlyAspValValTyrGly                              115120 125                                                                    MetIleArgThrAsnValCysAlaGluProGlyAspSerGlyGlyPro                              130135140                                                                     LeuTyrSerGlyThrArgAlaIleGlyLeuThrSerGlyGlySer Gly                             145150155160                                                                  AsnCysSerSerGlyGlyThrThrPhePheGlnProValThrGluAla                              165170 175                                                                    LeuValAlaTyrGlyValSerValTyr                                                   180185                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 181 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      IleAlaGlyGlyGluAlaIleThrThrGlyGlySerArgCysSerLeu                              151015                                                                        GlyPheAsnValSerValAsnGlyVa lAlaHisAlaLeuThrAlaGly                             202530                                                                        HisCysThrAsnIleSerAlaSerTrpSerIleGlyThrArgThrGly                              354 045                                                                       ThrSerPheProAsnAsnAspTyrGlyIleIleArgHisSerAsnPro                              505560                                                                        AlaAlaAlaAspGlyArgValTyrLeuTyrAsn GlySerTyrGlnAsp                             65707580                                                                      IleThrThrAlaGlyAsnAlaPheValGlyGlnAlaValGlnArgSer                              85 9095                                                                       GlySerThrThrGlyLeuArgSerGlySerValThrGlyLeuAsnAla                              100105110                                                                     ThrValAsnTyrGlySerSerG lyIleValTyrGlyMetIleGlnThr                             115120125                                                                     AsnValCysAlaGlnProGlyAspSerGlyGlySerLeuPheAlaGly                              130135 140                                                                    SerThrAlaLeuGlyLeuThrSerGlyGlySerGlyAsnCysArgThr                              145150155160                                                                  GlyGlyThrThrPheTyrGln ProValThrGluAlaLeuSerAlaTyr                             165170175                                                                     GlyAlaThrValLeu                                                               180                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 198 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AlaAsnIleValGlyGlyIleGluTyrSerIleAsnAsnAlaSerLeu                              1510 15                                                                       CysSerValGlyPheSerValThrArgGlyAlaThrLysGlyPheVal                              202530                                                                        ThrAlaGlyHisCysGlyThrValAsnAlaThrAla ArgIleGlyGly                             354045                                                                        AlaValValGlyThrPheAlaAlaArgValPheProGlyAsnAspArg                              505560                                                                        AlaTrpValSerLeuThrSerAlaGlnThrLeuLeuProArgValAla                              65707580                                                                      AsnGlySerSerPheValThrValArgGlySerThrG luAlaAlaVal                             859095                                                                        GlyAlaAlaValCysArgSerGlyArgThrThrGlyTyrGlnCysGly                              100105 110                                                                    ThrIleThrAlaLysHisValThrAlaAsnTyrAlaGluGlyAlaVal                              115120125                                                                     ArgGlyLeuThrGlnGlyAsnAlaCysMetGlyA rgGlyAspSerGly                             130135140                                                                     GlySerTrpIleThrSerAlaGlyGlnAlaGlnGlyValMetSerGly                              145150155 160                                                                 GlyAsnValGlnSerAsnGlyAsnAsnCysGlyIleProAlaSerGln                              165170175                                                                     ArgSerSerLeuPheGluArgLeuGln ProIleLeuSerGlnTyrGly                             180185190                                                                     LeuSerLeuValThrGly                                                            195                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GlyAsnSerGlyGlyAlaLeu                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GlyAspSerGlyGlyProLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D ) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      MetLysLysThrArgPheValLeuAsnSerIleAlaLeuGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CGCTCCACCAGCATTACCGCGG22                                                      (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ATCAATAACAGCATTATTGGT21                                                       (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        ( C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TAATGCAATTGCTGATAGTTC21                                                       (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      LysPhePhePheGlyAspArgPheAlaGluGln                                             1510                                                                      

What we claim is:
 1. An isolated and purified analog of Haemophilusinfluenzae Hin47 protein having a decreased protease activity which isless than about 10% of that of natural Hin47 protein, wherein at leastone amino acid of the natural Hin47 protein contributing to proteaseactivity and which is selected from the group consisting of amino acids91, 121 and 195 to 201 of natural Hin47 protein has been deleted orreplaced by a different amino acid to provide said reduced proteaseactivity.
 2. The analog of claim 1 having substantially the sameimmunogenic properties as natural Hin47 protein.
 3. The analog of claim1 wherein said at least one amino acid is Serine-197.
 4. The analog ofclaim 3 wherein Serine-197 is replaced by alanine.
 5. The analog ofclaim 1 wherein Histidine-91 is replaced by alanine, lysine or arginine.6. The analog of claim 1 wherein Asp-121 is replaced by alanine.
 7. Anisolated and purified nucleic acid molecule comprising a mutantHaemophilus influenzae hin47 gene coding an analog of Haemophilusinfluenzae Hin47 protein having a decreased protease activity which isless than about 10% of that of natural Hin47 protein, wherein at leastone codon of a wild-type hin47 gene encoding an amino acid contributingto protease activity which is the codon encoding an amino acid selectedfrom the group consisting of amino acids 91, 121 and 195 to 201, hasbeen deleted or replaced by a codon encoding a different amino acid toprovide the reduced protease activity.
 8. The nucleic acid molecule ofclaim 7 wherein said encoded analog has substantially the immunogenicproperties of natural Hin47 protein.
 9. The nucleic acid molecule ofclaim 7 wherein the at least one codon is that encoding Serine-197. 10.The nucleic acid molecule of claim 9 wherein the codon encodingSerine-197 is replaced by a codon encoding alanine.
 11. The nucleic acidmolecule of claim 7 wherein the codon encoding His-91 is replaced by acodon encoding alanine, lysine or arginine.
 12. The nucleic acidmolecule of claim 7 wherein the codon encoding Asp-121 is replaced by acodon encoding alanine or glutamic acid.
 13. The nucleic acid moleculeof claim 7 wherein said mutant gene is formed by site-directedmutagenesis of a wild-type hin47 gene.
 14. A recombinant plasmid adaptedfor transformation of a host cell comprising a plasmid vector into whichhas been inserted the nucleic acid molecule of claim 7..
 15. Therecombinant plasmid of claim 14 which is plasmid DS-1011-1-1(pT7/Hin47*) deposited under ATCC designation 75,845.
 16. A transformedcell containing the recombinant plasmid of claim
 14. 17. A method forproducing an analog of Haemophilus influenzae Hin47 protein having areduced protease activity which is less than about 10% of natural Hin47protein, which comprises:identifying at least one amino acid residue ofHin47 protein which contributes to protease activity thereof; effectingsite-directed mutagenesis of the hin47 gene to remove or replace anucleotide sequence encoding at least one amino acid selected from thegroup consisting of amino acids 91, 121 and 195 to 201 of the naturalHin47 protein and to produce a mutated hin47 gene; introducing themutated hin47 gene into a cell to produce a transformed cell; andgrowing the transformed cell to produce the Hin47 analog.
 18. The methodof claim 17 wherein said at least one amino acid is Serine-197.
 19. Themethod of claim 18 wherein Serine-197 is replaced by alanine.
 20. Themethod of claim 17 wherein Histidine-91 is replaced by alanine, lysineor arginine.
 21. The method of claim 17 wherein Asp-121 is replaced byalanine or glutamic acid.
 22. The method of claim 17 wherein saidintroduction of the mutated hin47 gene produces a transformed cell inwhich the mutated hin47 gene is under control of the T7 promoter, andsaid growing of said transformed cell and expression of the Hin47 analogby said T7 promoter is effected by culturing in an inducingconcentration of lactose.
 23. The method of claim 22 wherein saidintroduction of the mutated hin47 gene is effected by transforming saidcell with the recombinant plasmid DS-1011-1-1 (pT7/Hin47*) depositedunder ATCC designation 75,845.
 24. A method of providing an isolated andpurified analog of Haemophilus influenzae Hin47 protein having a reducedprotease activity which is less than about 10% of natural Hin47 protein,which comprises:identifying at least one amino acid residue of Hin47protein which contributes to protease activity thereof; effectingsite-directed mutagenesis of the hin47 gene to remove or replace anucleotide sequence encoding at least one amino acid selected from thegroup consisting of amino acids 91, 121 and 195 to 201 of the naturalHin47 protein and to produce a mutated hin47 gene; introducing themutated hin47 gene into a cell to produce a transformed cell; growingthe transformed cell to produce grown transformed cells harbouringinclusion bodies containing the Hin47 analog; disrupting said growntransformed cells to produce supernatant and said inclusion bodies;solubilizing said inclusion bodies to produce a solution containingHin47 analog; chromatographically purifying said Hin47 analog from saidsolution free from cell debris; and isolating said purified Hin47analog.
 25. The method of claim 24 wherein said introduction of themutated hin47 gene is under control of the T7 promoter, and the growingof said transformed cell and expression of the Hin47 analog by said T7promoter is effected by culturing in an inducing concentration oflactose.
 26. A chimeric molecule, comprising an analog as claimed inclaim 1 linked to a polypeptide, protein or a polysaccharide.